A striking feature of the biology of Haemophilus influenzae is that these organisms can be distinguished from nearly all other facultative, gram-negative bacteria by their absolute requirement for heme for aerobic growth. During the current funding period, the PI proved his hypothesis that H. influenzae has evolved distinct mechanisms for acquiring free and protein-bound forms of heme; the latter are the heme compounds found in vivo. The objective of the proposed research project is the elucidation of the molecular basis for heme acquisition by this pathogen, with particular emphasis on how nontypeable strains of H. influenzae (NTHI) obtain heme from the serum proteins hemopexin and haptoglobin which bind heme and hemoglobin, respectively. The first Specific Aim will investigate the genetic organization of the hxuCBA gene cluster and determine the precise physiologic function of the HxuC protein which appears to be a TonB-dependent outer membrane protein and may be involved in the utilization of heme: hemopexin. The second Specific Aim will investigate the parameters involved in the binding of heme: hemopexin complexes by the soluble HxuA protein. The PI has discovered that the soluble l00 kDa HxuA protein functions in a siderophore-like manner, binding heme: hemopexin complexes and making them available to the H. influenzae cell for utilization of the heme moiety. He will also identify the bacterial receptor for HxuA/heme: hemopexin complexes. The third Specific Aim will involve identification of the complete complement of NTHI outer membrane receptors for hemoglobin: haptoglobin and hemoglobin. Analysis of a NTHI gene (hhuA) encoding a hemoglobin:haptoglobin-binding outer membrane protein indicates that there may exist a family of structurally related H. influenzae proteins that bind hemoglobin or hemoglobin: haptoglobin. Which of these different heme acquisition systems are essential for survival and growth of NTHI in vivo will be determined in the fourth Specific Aim.